Characterisation of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2'' of methicillin-resistant Staphylococcus aureus

The additional penicillin-binding protein (PBP) 2' that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been purified by affinity chromatography using monoclonal antibodies. Monoclonal antibody 1/423.10.351 reacted in ELISA with detergent extracts of membranes from resistant organisms, but not in immunoblots with PBP 2' separated by SDS-PAGE. Immunoprecipitation experiments showed that antibody 1/423.10.351 reacted with PBP 2' in detergent extracts. The latter antibody, covalently coupled to protein A-Sepharose through the Fc region, served as an affinity matrix to purify PBP 2'. The PBP was detected in immunoblots using a second monoclonal antibody, 2/401.43. Conjugation of this antibody with alkaline phosphatase afforded more rapid detection of PBP 2' for the immunological detection of PBP 2' both in affinity-purified fractions and in resistant strains.     

Methicillin-resistant strains of Staphylococcus aureus; presence of identical additional penicillin-binding protein in all strains examined

Abstract The methicillin-resistant strain of Staphylococcus aureus MR-1 previously reported to possess a penicillin-binding protein 3 (PBP 3) with a decreased affinity for β-lactam antibiotics was re-examined and, in common with other resistant strains, found to contain an additional PBP (PBP 2'). Expression of the additional protein, which has a very low affinity for β-lactams, was not influenced by temperature or osmolarity of the medium in contrast with strains examined previously. It was the only PBP still available to bind radioactive β-lactams and therefore still active enzymically when strain MR-1 was grown in the presence of concentrations of β-lactam antibiotics sufficient to kill sensitive strains of S. aureus . Penicillin-peptides derived by partial proteolysis of PBP 2'-penicillin complexes of MR-1 and 3 other methicillin-resistant strains appeared to be identical and different from the penicillin-peptides derived from PBP 1, PBP 2 and PBP 3, each of which gave rise to a unique series of peptides containing covalently-bound penicillin.     

Methicillin resistance in Staphylococcus epidermidis. Relationship between the additional penicillin-binding protein and an attachment transpeptidase

The penicillin-binding proteins (PBP) of a methicillin-resistant strain of Staphylococcus epidermidis, 100,604 p+m+ and a non-isogenic sensitive strain, p-m- were characterised. The presence of a novel PBP, produced by the methicillin-resistant strain of S. epidermidis, with an Mr identical to that of PBP2' in Staphylococcus aureus 13,136 p-m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30 degrees C, in media containing beta-lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non-growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100,604 p+m+ and from PBP2' in S. aureus 13,136 p+m+ lends support to the theory that the additional DNA encoding PBP2' in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice-versa, indicated that there was a strong correlation between the presence of PBP2' and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of beta-lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of beta-lactam antibiotics at either 40 degrees C or 30 degrees C suggests that there is little relationship between the production of this enzyme and of PBP2' other than the fact that beta-lactam antibiotics are common inducers of both.     

Effects of tungstosilicate on strains of methicillin-resistant Staphylococcus aureus with unique resistant mechanisms

In a previous study, it was found that polyoxotungstates such as undecatungstosilicate (SiW11) greatly sensitized strains of methicillin-resistant Staphylococcus aureus (MRSA) to beta-lactams. In this report, the effects of SiW11 on several MRSA strains with unique resistant mechanisms were studied. SiW11 was still effective to MRSA mutants with higher beta-lactam resistance due to reduced cell-lytic activity. Since the antimicrobial effect of TOC-39 (a cephem antibiotic with strong affinity to penicillin-binding protein (PBP) 2') was not strongly enhanced in any case, it was confirmed that the sensitizing effect of SiW11 is due to reduced expression of PBP2'. However, the sensitizing effect of SiW11 was relatively weak in MRSA strains with lowered susceptibility to glycopeptide antibiotics. A certain resistant mechanism other than the mecA-PBP2' system worked in such a strain. Interestingly, an MRSA mutant with the Eagle-type resistance was dramatically sensitized. This result suggests that SiW11 has another site of action besides reducing the expression of PBP2'.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.     

Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.     

Mechanistic basis for the action of new cephalosporin antibiotics effective against methicillin- and vancomycin-resistant Staphylococcus aureus

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created challenges in treatment of nosocomial infections. The recent clinical emergence of vancomycin-resistant MRSA is a new disconcerting chapter in the evolution of these strains. S. aureus normally produces four PBPs, which are susceptible to modification by beta-lactam antibiotics, an event that leads to bacterial death. The gene product of mecA from MRSA is a penicillin-binding protein (PBP) designated PBP 2a. PBP 2a is refractory to the action of all commercially available beta-lactam antibiotics. Furthermore, PBP 2a is capable of taking over the functions of the other PBPs of S. aureus in the face of the challenge by beta-lactam antibiotics. Three cephalosporins (compounds 1-3) have been studied herein, which show antibacterial activities against MRSA, including the clinically important vancomycin-resistant strains. These cephalosporins exhibit substantially smaller dissociation constants for the preacylation complex compared with the case of typical cephalosporins, but their pseudo-second-order rate constants for encounter with PBP 2a (k(2)/K(s)) are not very large (< or =200 m(-1) s(-1)). It is documented herein that these cephalosporins facilitate a conformational change in PBP 2a, a process that is enhanced in the presence of a synthetic surrogate for cell wall, resulting in increases in the k(2)/K(s) parameter and in more facile enzyme inhibition. These findings argue that the novel cephalosporins are able to co-opt interactions between PBP 2a and the cell wall in gaining access to the active site in the inhibition process, a set of events that leads to effective inhibition of PBP 2a and the attendant killing of the MRSA strains.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.     

Fosfomycin enhances the expression of penicillin-binding protein 2 in methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains

The effects of fosfomycin on penicillin-binding proteins (PBPs) were studied on the methicillin-resistant Staphylococcus aureus strain CIP (Collection de l'Institut Pasteur, Paris, France) 65-25 and on a methicillin-susceptible S. aureus strain CIP 65-6. The combinations of fosfomycin and oxacillin were synergistic, additive or antagonistic, depending on antibiotic concentrations. Fosfomycin induced modifications of the PBP profile of the two strains studied. In particular, it increased the expression of PBP2. This suggested that this protein is inducible; the only PBP not affected by fosfomycin was PBP3.     

Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction.

Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.     

Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.     

Impact of sar and agr on methicillin resistance in Staphylococcus aureus

Abstract The global regulators agr and sar control expression of cell wall and extracellular proteins. Inactivation of either sar and/or agr in a typical heterogeneously methicillin-resistant Staphylococcus aureus resulted in a small but reproducible decrease in the number of cells in the subpopulation expressing high methicillin resistance. The amount of low affinity penicillin-binding protein PBP2', the prerequisite for methicillin resistance, was apparently not affected, however, a reduction in PBP1 and PBP3 production was observed, suggesting that these resident PBPs of the cells might be involved somehow together with PBP2' in high level methicillin resistance.     

Cell wall-affecting antibiotics induce expression of a novel gene, drp35, in Staphylococcus aureus.

A novel gene, drp35, of Staphylococcus aureus, which was inducible especially with cell wall-affecting antibiotics, has been cloned. Analysis of differential hybridization with mRNAs enhanced in the presence of beta-lactams resulted in two positive clones that harbored a new gene encoding a 35,845-Da protein (Drp35) and the penicillin-binding protein 2 (PBP2). Immunoblot analysis revealed that the Drp35 protein band was evidently enhanced after 30 min in the presence of beta-lactams. The Drp35 expression was also enhanced with not only beta-lactams, but also vancomycin, bacitracin, and fosfomycin. Homology search revealed that Drp35 was a new protein. Our results revealed that it was specific in S. aureus and respondent to these agents in both methicillin-resistant and -sensitive strains of S. aureus.     

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.     

femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) with Antibodies against Synthetic Peptides Derived from Penicillin-Binding Protein 2′

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2′ (PBP2′) of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2′ of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2′ was developed using these antibodies. The method could detect PBP2′ extracted from as few as 3 × 10⁴ cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.     

Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus.

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 micrograms/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 micrograms/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants. Insertion of Tn918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.     

Reduction of the Peptidoglycan Crosslinking Causes a Decrease in Stiffness of the Staphylococcus aureus Cell Envelope

We have used atomic-force microscopy (AFM) to probe the effect of peptidoglycan crosslinking reduction on the elasticity of the Staphylococcus aureus cell wall, which is of particular interest as a target for antimicrobial chemotherapy. Penicillin-binding protein 4 (PBP4) is a nonessential transpeptidase, required for the high levels of peptidoglycan crosslinking characteristic of S. aureus. Importantly, this protein is essential for β-lactam resistance in community-acquired, methicillin-resistant S. aureus (MRSA) strains but not in hospital-acquired MRSA strains. Using AFM in a new mode for recording force/distance curves, we observed that the absence of PBP4, and the concomitant reduction of the peptidoglycan crosslinking, resulted in a reduction in stiffness of the S. aureus cell wall. Importantly, the reduction in cell wall stiffness in the absence of PBP4 was observed both in community-acquired and hospital-acquired MRSA strains, indicating that high levels of peptidoglycan crosslinking modulate the overall structure and mechanical properties of the S. aureus cell envelope in both types of clinically relevant strains. Additionally, we were able to show that the applied method enables the separation of cell wall properties and turgor pressure.